microRNAs (miRNAs) are central regulators of a wide array of biological processes including cancer, development, neurodegenerative and metabolic diseases and viral infection. Yet, the details of their molecular mechanisms remain controversial. In the course of our studies on cellular quiescence in human cells, we discovered that the let-7 miRNA likely regulates not only genes with recognition sequences in their 3'UTRs but also genes with recognition sequences in their coding regions. We further discovered evidence for mechanistic differences between the targeting of 3'UTR versus coding region recognition sites. Our results in combination with published data also raise the possibility of target-specificity in miRNA mechanisms in the context of quiescence. We propose here a combined computational and experimental approach to test our hypothesis that miRNAs have distinct mechanisms of action depending upon the interaction between the miRNA and the target. We expect that the results will be valuable for predicting physiologically relevant targets of miRNAs and for designing maximally effective miRNA therapeutics. In our first aim, we will use a combination of comparative genomics and reporter assays to determine whether there are multiple distinct mechanisms by which miRNAs target different portions of transcripts. In Specific aims #2 - #4, we will test our hypothesis that there are transcript-specific mechanisms that govern whether miRNAs regulate targets via transcript degradation or translation. We will analyze cells transfected with one of several miRNAs by performing microarray analysis on total transcripts and polysome-associated transcripts. These data will allow us to identify characteristics of miRNA-target interactions that result in transcript degradation and those that affect translation initiation. In specific aim #3, we will extend these experiments to test our hypothesis that there is a target-specific switch in miRNA activity from repression to activation as cells become quiescent. Finally, in specific aim #4, we will extend our analysis to endogenous miRNAs by monitoring the impact of anti-miRs, miRNA inhibitors, on total and polysome-associated transcripts. Our expectation is that deciphering the mechanisms of miRNA function, including the basis for specificity for miRNA-target interactions, will provide clarity to the field of miRNA research. The findings, which will be made available through our website, will also advance many related fields as researchers studying a wide array of biological processes and disease states are better able to associate miRNAs with their targets and functions. PUBLIC HEALTH RELEVANCE: While miRNAs are clearly central players in a wide array of biological processes including cancer, development, neurodegenerative and metabolic diseases and viral infection, linking miRNAs with the specific molecules that they regulate has been a challenge. We propose here a combined computational and experimental approach to elucidate the mechanisms of miRNA-target interactions for many different miRNA- target pairings. We anticipate that these findings will be of great value to researchers in a wide range of fields working to identify miRNAs targets, and those designing miRNAs as therapeutics.